Individual Active Ingredients Research Articles

Towards a comprehensive methodology for ecotoxicological assessment: Prioritizing plant protection products for mixture testing in edge-of-field surface waterbodies

Pesticide applications in agriculture result in complex mixtures of Plant Protection Products (PPPs) in the environment. The ecotoxicological effects of these mixtures can occur at concentrations considered safe for individual chemicals, indicating potential risks underestimated by current regulatory assessments focused on individual active ingredients. To address this challenge, our study introduces a methodology for identifying priority PPPs for formulating mixtures, enabling further ecotoxicological testing in water and sediment compartments of edge-of-field surface water bodies, targeting pelagic and benthic organisms. This methodology was primarily based on the actual quantification of PPPs present in these compartments from selected case study sites (CSSs) in Europe and Argentina (11 and 4 for water and sediments, respectively). A conceptual framework was developed that discriminates and selects concerning PPPs based on their individual risk quotient and frequency of occurrence in each CSS, drawing upon two EU regulatory risk assessment approaches, i.e., the general approach under REACH for any environmental contaminant of concern – the European Chemicals Agency (ECHA) approach; and that specifically focusing on PPPs – the European Food Safety Authority (EFSA) approach. Irrespective of whether the focus is on water or sediments, the study revealed disparities in PPP rankings depending on the approach used to identify PPPs of concern, with the ECHA approach being more conservative than the EFSA approach. Despite this, the EFSA approach follows a more standardized assessment factor definition strategy, potentially allowing avoidance of risk overestimation, as well as resulting in a more balanced representation of different PPP classes for subsequent mixtures testing. Overall, the methodological development reported herein, along with the inconsistencies found when comparing different regulatory approaches to assess the risk of environmental contaminants, highlight the need for further discussion on the most appropriate directions towards the standardization of the regulatory risk assessment of PPP mixtures.

Application Characteristics and Patient Preference of Triple-Combination vs Layered Topicals for Acne: Split-Face Study.

Although triple-combination therapies for acne are generally more efficacious than dual-combinations or topical monotherapy, this benefit may be offset by reduced adherence to a complicated treatment regimen. Clindamycin phosphate 1.2%/adapalene 0.15%/benzoyl peroxide 3.1% (CAB; Cabtreo®, Ortho Dermatologics) gel is the first triple-combination topical approved for the treatment of acne. By delivering multiple active ingredients as a fixed-dose combination, CAB gel may improve ease of use, which can benefit both treatment adherence and efficacy. The objective of this study was to compare the application characteristics of CAB gel with the layered application of its 3 individual active ingredients. In this split-face study, adults with acne-prone skin (N=25), self-applied CAB gel (0.3 cc) to 1 side of the face and layered benzoyl peroxide cream, adapalene gel, and clindamycin gel (0.1 cc each) on the opposite side. CAB and clindamycin gels were compounded with pyranine, which fluoresces under blue light. Photos taken under blue light were used to assess the uniformity of product application, and participants rated the evenness, speed, and ease of the 2 application regimens, as well as overall preference. Investigator-assessed evenness of application favored CAB gel over layered application in 100% of participants. All participants rated the application of CAB gel as more uniform, easier, and faster. Most (96%) preferred CAB gel for use at home. Fixed-dose CAB gel was applied more evenly than separate application of its 3 active ingredients. By addressing 3 of the main acne pathogenic pathways in a single, easy-to-apply formulation, CAB gel may improve the efficacy of and adherence to acne treatment. J Drugs Dermatol. 2024;23(10):857-861. doi:10.36849/JDD.8430.

A combination of carbonates and Opuntia ficus-indica extract protects esophageal cells against simulated acidic and non-acidic reflux in vitro

Buffering of stomach acid by antacids is a well-established symptomatic therapy for heartburn. In addition, preparations from prickly pear (Opuntia ficus-indica) have been shown to reduce tissue damage in experimental gastritis models and to attenuate gastrointestinal discomfort in patients. Both active principles have been included in a fixed-combination product for symptomatic treatment of heartburn containing carbonate antacids (CaCO3 and MgCO3) and an extract from Opuntia ficus-indica cladodes. The aim of the study was to characterize the acid neutralization and esophageal cell protective activities of the product and its individual active ingredients in a set of in vitro assays. Acid neutralization was assessed in a simulated stomach model. Protective activity of individual constituents and in combination was analyzed in an esophageal cell line (COLO-680 N) exposed to low pH and deoxycholic acid to simulate acidic and non-acidic reflux challenge. The combination product protected cells against low pH mediated cytotoxicity via acid neutralization by carbonates. Opuntia extract itself and the combination product attenuated bile acid-induced cell irritation as measured by reduced release of proinflammatory interleukin-6 and -8. In conclusion, addition of Opuntia extract to a mineral antacid provides dual protection against acidic and non-acidic simulated reflux challenge.

Contact exposure of honey bees and social stingless bees to fungicide sprayed on cotton and soybean in a controlled field simulation system

AbstractBees can be exposed to pesticides when visiting crops or plants in adjacent areas affected by spray drift. Among pesticide categories, fungicides tend to be considered relatively safe, though they also can negatively affect pollinators. Most evidence of damage by fungicides to bees comes from laboratory tests; there is little information concerning contamination levels in the field. We examined exposure of honey bees (Apis mellifera L.) (Hymenoptera: Apidae) and a common Brazilian native species of social stingless bees (Scaptotrigona postica Latreille; Hymenoptera: Apidae), which is about a third the size of a honey bee, to a commercial fungicide (Fox Xpro), with three active ingredients (trifloxystrobin, bixafen, and prothioconazole), applied to crops they often visit according to label directions. A spraying apparatus mounted on tracks in a laboratory spray room was used to simulate field conditions. Soybean and cotton plants grown in pots were transferred to the spray room when the plants were in flower. Anaesthetized bees were attached with insect pins at the top and middle of the plants, on leaves and flowers. The fungicide was applied using fine or coarse droplets. The amounts of the individual active ingredients absorbed by bees were then quantified. Concentrations of trifloxystrobin were highest in both honey bees and stingless bees, followed by bixafen, and then prothioconazole, which was detected in the bees at much lower levels. Overall, bees at the top of the plants and those sprayed with fine droplets absorbed more pesticide. As a function of body mass, the stingless bees were more heavily contaminated than the larger honey bees. Tests using spraying systems that simulate field conditions can better estimate the actual doses that contaminate bees to help determine the impact of fungicides and other pesticides applied to crops.

Triple-Combination Clindamycin Phosphate 1.2%/Adapalene 0.15%/Benzoyl Peroxide 3.1% Gel for Acne: Clinical Efficacy and Application Characteristics

Background: Triple-combination therapies for acne including an antibiotic, topical retinoid, and benzoyl peroxide (BPO) are among the most effective, with meta-analyses demonstrating greater efficacy with triple-combinations than dual-combinations or topical monotherapy. However, this benefit may be offset by reduced adherence to a complicated treatment regimen. Here, the clinical efficacy of fixed-dose clindamycin phosphate 1.2%/adapalene 0.15%/BPO 3.1% (CAB) gel is reviewed, and the ease of CAB application is compared with the layered application of its individual active ingredients.
 Methods: In a phase 2 (N=741) and two phase 3 (N=183; N=180), double-blind, randomized, 12-week studies, participants aged ≥9 years with moderate-to-severe acne were randomized to receive once-daily CAB or vehicle; the phase 2 study also included treatment arms containing dyad gels (BPO/adapalene; clindamycin phosphate/BPO; clindamycin phosphate/adapalene). Efficacy endpoints included treatment success (percentage of participants achieving ≥2-grade reduction from baseline in Evaluator’s Global Severity Score and clear/almost clear skin) and reductions from baseline in inflammatory (IL) and noninflammatory lesions (NIL). In a split-face study of adults with acne-prone skin (N=25), participant-application of CAB (0.3 cc) was compared to sequential, layered application of benzoyl peroxide cream, adapalene gel, and clindamycin gel (0.1 cc each). IDP-126 and clindamycin gels were compounded with pyranine, which fluoresces under blue light; photos were taken under blue light to assess evenness of product application.
 Results: In all three clinical studies at week 12, half of CAB-treated participants achieved treatment success (range: 49.6%-52.5%), significantly greater than with vehicle (8.1%-24.9%; P<0.01, all) or dyads (phase 2 study only; 27.8%-30.5%; P≤0.001, all). Reductions from baseline in both IL and NIL were also significantly greater for CAB vs vehicle (range, IL: 75.7%-80.1% vs 50.4%-59.6%; NIL: 71.0%-73.3% vs 45.8%-49.0%; P<0.001, all) and dyads (IL: 64.0%-69.2%; NIL: 58.7%-61.1%; P<0.01, all vs IDP-126). In the split-face study, 100% of Investigator and participant assessments of evenness of application favored CAB over the three layered products. In addition, all participants rated CAB as both easier and faster to apply, and most (96%) preferred CAB for use at home.
 Conclusions: Fixed-dose CAB gel applied more evenly than separate application of its three active ingredients, and demonstrated significantly greater efficacy in the treatment of moderate-to-severe acne than dyad gels or vehicle. By addressing three of the main acne pathogenic pathways in a single, easy-to-apply formulation, CAB may improve efficacy of and adherence to acne treatment.
 Funding: Ortho Dermatologics

Efficacy of Unregulated Minimum Risk Products to Kill and Repel Ticks.

Human-biting ticks threaten public health in the United States. Registration by the Environmental Protection Agency of products to kill host-seeking ticks or repel ticks contacting humans is indicative of their safety and effectiveness. Unregulated minimum risk products, exempt from Environmental Protection Agency registration and often based on botanical oils, are proliferating in the marketplace, but there is concern about their effectiveness to kill and repel ticks. Evaluations of such products are limited in the published literature. A review showed considerable variability among minimum risk products to kill host-seeking blacklegged ticks, with effectiveness similar to chemical pesticide products for some minimum risk products but minimal impact on the ticks for other products. Evaluations of minimum risk tick repellents have typically focused on individual active ingredients rather than formulated products, which often combine multiple active ingredients. Consumers should be aware that effectiveness to kill and repel ticks can differ among unregulated minimum risk products.

Therapeutic advantages of the combined use of closantel and moxidectin in lambs parasitized with resistant gastrointestinal nematodes

The serious widespread development of nematode resistance has motivated the use of combined anthelmintic formulations. However, the advantages/disadvantages of the combined use of anthelmintics require further scientific characterization. The goals of the current trial were a) to characterize the pharmacokinetics of closantel (CLO) and moxidectin (MXD) administered both subcutaneously (sc) and orally either separately or co-administered (CLO + MXD) to lambs; b) to compare the nematodicidal activity of both molecules given individually or co-administered to lambs infected with resistant nematodes. Seventy (70) Corriedale lambs naturally infected with multiple resistant gastrointestinal nematodes were involved in the pharmacokinetic and efficacy trials. The animals were allocated into six groups (n = 10) and treated with either CLO, MXD, or with the CLO + MXD combined formulation by both the oral and sc routes. Additionally, an untreated control group (n = 10) was included for the efficacy trial. The efficacy was estimated by the faecal egg count reduction test (FECRT). Higher systemic exposure of both CLO and MXD was observed after the sc compared to the oral administration in lambs. The combined administration of CLO + MXD did not markedly alter their disposition kinetics. At 13 days post-treatment, the administration of both molecules as a single active principle reached efficacy levels ranging between 80% (MXDoral), 84% (CLOoral), 85% (CLOsc), and 92% (MXDsc). The combined oral and sc treatments reached 99% efficacy. No adverse effects were observed after the combined treatment of CLO + MXD, and their co-administration did not show any adverse pharmacokinetic interaction. The combined effect of CLO + MXD successfully restored the maximum efficacy levels, which were not reached by the individual active ingredients.

Exposure to pesticides and risk of Hodgkin lymphoma in an international consortium of agricultural cohorts (AGRICOH)

PurposeSome pesticides may increase the risk of certain lymphoid malignancies, but few studies have examined Hodgkin lymphoma (HL). In this exploratory study, we examined associations between agricultural use of 22 individual active ingredients and 13 chemical groups and HL incidence.MethodsWe used data from three agricultural cohorts participating in the AGRICOH consortium: the French Agriculture and Cancer Cohort (2005–2009), Cancer in the Norwegian Agricultural Population (1993–2011), and the US Agricultural Health Study (1993–2011). Lifetime pesticide use was estimated from crop-exposure matrices or self-report. Cohort-specific covariate-adjusted overall and age-specific (< 40 or ≥ 40 years) hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated using Cox regression and combined using random effects meta-analysis.ResultsAmong 316 270 farmers (75% male) accumulating 3 574 815 person-years at risk, 91 incident cases of HL occurred. We did not observe statistically significant associations for any of the active ingredients or chemical groups studied. The highest risks of HL overall were observed for the pyrethroids deltamethrin (meta-HR = 1.86, 95% CI 0.76–4.52) and esfenvalerate (1.86, 0.78–4.43), and inverse associations of similar magnitude were observed for parathion and glyphosate. Risk of HL at ≥ 40 years of age was highest for ever-use of dicamba (2.04, 0.93–4.50) and lowest for glyphosate (0.46, 0.20–1.07).ConclusionWe report the largest prospective investigation of these associations. Nonetheless, low statistical power, a mixture of histological subtypes and a lack of information on tumour EBV status complicate the interpretability of the results. Most HL cases occurred at older ages, thus we could not explore associations with adolescent or young adult HL. Furthermore, estimates may be attenuated due to non-differential exposure misclassification. Future work should aim to extend follow-up and refine both exposure and outcome classification.

Recombinant glutathione transferases from flufenacet-resistant black-grass (Alopecurus myosuroides Huds.) form different flufenacet metabolites and differ in their interaction with pre- and post-emergence herbicides.

Black-grass (Alopecurus myosuroides Huds.) has become a problematic weed in cereals in Europe. Besides resistance to post-emergent herbicides becoming increasingly widespread, enhanced metabolism of inhibitors of the synthesis of very-long-chain fatty acids (VLCFAs), such as flufenacet, is evolving. Yet, cross-resistance patterns and evolution of this resistance remains poorly understood. The cDNA sequences of five glutathione transferases (GSTs) upregulated in flufenacet resistant black-grass were identified and used for recombinant protein expression. Moderate to slow detoxification of flufenacet was verified for all candidate GSTs expressed in E. coli, and the most active protein produced flufenacet-alcohol instead of a glutathione conjugate, in the presence of reduced glutathione (GSH). Moreover, cross-resistance to other VLCFA-inhibitors e.g., acetochlor and pyroxasulfone and the ACCase inhibitor fenoxaprop was verified in vitro. Various other herbicides of different modes of action including VLCFA-inhibitors were not detoxified by the candidate GSTs. As several in planta upregulated GSTs detoxified flufenacet in vitro, the shift in sensitivity observed in black-grass populations, is likely a result of an additive effect. The polygenic character and the relatively low turnover rate of the individual GSTs may explain the slow evolution of flufenacet resistance. In addition, flufenacet resistance was accompanied by cross-resistance with some, but not all, herbicides of the same mode of action, and furthermore to the ACCase inhibitor fenoxaprop-ethyl. Hence, not only the rotation of herbicide modes of action, but also of individual active ingredients is important for resistance management. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Preciseness, rather than simplicity, is required to assess pesticide reduction strategies: Findings from rice production in Japan

Pesticide reduction is given high priority in the worldwide sustainability agenda. The reduction of pesticide impacts, rather than the reduction of application rates, has become a common criterion for monitoring policy progress. However, simplicity—an essential requirement in improving the applicability of pesticide impact assessment—may distort the accuracy of the evaluation and therefore prevent effective pesticide reduction. Here, we present contrasting results that underscore how the selection of evaluation methods that differ in simplicity affects the assessment results of pesticide reduction strategies. Briefly, we analysed the impact of conversion from conventional to low-input management adopting both a simplified linear-based method and a precise method that includes newly calculated nonlinear approach-based characterization factors for 109 active ingredients (AIs). The two methods were then used to estimate the freshwater ecotoxicity impact of eight rice farms in Japan where both conventional pesticide application and pesticide reduction strategies are practiced. The results show that the simplified method generated anomalies at the farm level through overestimation and underestimation of the individual AI impacts. Patterns that contributed to extreme changes of impact at the farm level were also identified. These findings suggest a strong need for a precise evaluation method for effectively monitoring policy progress at the farm level.

Plasma pharmacokinetics of tigolaner, emodepside, and praziquantel following topical administration of a combination product (Felpreva®) and of intravenous administration of the individual active ingredients in cats

Felpreva® for cats contains the new acaricidal/insecticidal active ingredient tigolaner in a fixed combination with the nematocidal and cestocidal compounds emodepside and praziquantel, respectively. The plasma pharmacokinetics of tigolaner, emodepside, and praziquantel were evaluated in clinically healthy cats following topical (spot-on) treatment as fixed combination Felpreva®. For the determination of bioavailability intravenous administration of single active ingredients was also performed. After a single topical administration of Felpreva® using the target dose volume of 0.148 ​ml/kg to cats, tigolaner reached mean peak concentrations of 1352 ​μg/l with a Tmax of 12 days and a mean half-life of 24 days. Simulation of repetitive topical administration every 91 days indicates only a low risk of accumulation after reaching steady state within two to three administrations. The volume of distribution calculated after intravenous dosing was 4 ​l/kg and plasma clearance was low with 0.005 ​l/h/kg. Overall plasma exposure was 1566 ​mg∗h/l after topical administration, providing an absolute bioavailability of 57%. Tigolaner was mainly cleared via the faeces (54% within 28 days), renal clearance was neglectable (< 0.5% within 28 days). Emodepside and praziquantel showed mean peak concentrations of 44 ​μg/l and 48 ​μg/l (reached after 1.5 days and 5 ​h, respectively). Overall plasma exposures were 20.6 and 3.69 ​mg∗h/l, respectively. The elimination half-life was 14.5 days for emodepside and 10 days for praziquantel after topical administration. After topical administration of Felpreva® using 2.5× and 5× dose multiples an almost proportional increase of plasma exposure was observed for all three active ingredients. With the addition of tigolaner, Felpreva® combines the established pharmacokinetic (PK) characteristics of emodepside and praziquantel contained in Profender® spot-on for cats with the favourable PK of tigolaner suitable for a 3-months protection against fleas and ticks.

UV-responsive fluorescent behavior of pharmaceuticals assessed by UV-induced fingerprint spectroscopy (UV-IFS)

UV-induced fingerprint spectroscopy (UV-IFS), a new tool in a toolbox of analytical methods, is a powerful technique registering molecule-specific changes of fluorescence induced by UV irradiation. Analysis of fluorescence spectra of a sample prior and after UV irradiation enables an identification of a sample of a drug or pharmaceutics based on a comparison with signals of known standards. Moreover, UV-IFS uncovers the presence of undesired contaminations or intentional changes of the composition. Herein, we employ UV-IFS for qualitative as well as quantitative analysis of common medicines including analgesic/antipyretic (Acetaminophen), antihistamines (Loratadine and Desloratadine), and phosphodiesterase type 5 inhibitors (Tadalafil and Sildenafil citrate). UV irradiation (λem = 254 nm) for 2 – 10 min induced significant changes of fluorescence of the studied samples and according to the unique patterns, the quality and quantity were evaluated. Limits of detection for individual active ingredients were calculated as follows: Acetaminophen = 0.1 µg·mL−1, Loratadine = 0.1 μg·mL−1, Desloratadine = 0.01 µg·mL−1, Tadalafil = 0.04 µg·mL−1 and Sildenafil = 0.2 µg·mL−1. Moreover, genuine and fake CIALIS, VIAGRA and KAMAGRA tablets were reliably identified.

Comparative Toxicity of Herbicide Active Ingredients, Safener Additives, and Commercial Formulations to the Nontarget Alga Raphidocelis Subcapitata.

Chloroacetanilide herbicides are used worldwide to control weeds that affect crops such as corn, soybeans, and cotton. These herbicides are frequently paired with a "safener," which prevents herbicidal damage to the crop without diminishing weed control. Formulated herbicide products that include safeners and other ingredients are infrequently assessed for toxicity. Our goal was to understand the potential toxicity of safeners and herbicide + safener formulations relative to the toxicity of associated active ingredients. We quantified the concentration of safeners in commercially available formulations and tested effects on nontarget algae, Raphidocelis subcapitata, when exposed to individual herbicide active ingredients, safeners, and commercial formulations. The median effective concentrations (EC50s) causing 50% reduction in population growth for the herbicide active ingredients S-metolachlor and acetochlor were 0.046 and 0.003 ppm, respectively. The safeners benoxacor, AD-67, furilazole, and dichlormid were all substantially less toxic than the herbicides and were not toxic at environmentally relevant concentrations. The commercial formulations Dual II Magnum®, Me-Too-Lachlor II®, Harness®, and Surpass EC® all resulted in EC50 values that fell within the 95% confidence interval of the associated active ingredient herbicide. Interestingly, a significant increase in cell size was observed when algae were exposed to all the formulations, herbicides (acetochlor and S-metolachlor), and safener (dichlormid). The safener furilazole caused a significant decrease in cell size, whereas benoxacor and AD-67 had no observed effect on algae cell size. Significant algae cell size effects all occurred at or above the EC50 concentrations for each chemical, suggesting that other morphological effects may be occurring. Importantly, safeners in commercial formulations appeared not to impact toxicity to R. subcapitata compared with the active ingredient alone.Environ Toxicol Chem 2022;41:1466-1476. © 2022 SETAC.

Improvement of fire protection of fabrics with intumescent coatings

Based on the analysis of literature sources, the article finds that today it is difficult to imagine the protection of human lives from fire without fire-retardant coatings on textiles. Precisely defined standards allow to determine the requirements for textile materials that have fire protection. It is hardly possible to prevent the inflammation of textiles due to their physical parameters (melting point, flash point). However, it is possible to extend the time for people to escape to start rescue operations by using appropriate fire protection systems or a combination of individual active ingredients. Reducing flammability and developing flammable and combustible materials is one of the main ways to prevent fires and solve the problem of expanding the scope of these materials. Treatment with flame retardants significantly affects the spread of flames, can significantly reduce the ability to smoke and heat. Therefore, research aimed at determining the patterns of fire protection paths used for the manufacture of textiles is relevant. One of such promising areas is the study of the formation of a layer of foam cone in the fire protection of the fabric with intumescent coatings. Experimental studies to determine the fire-hazardous properties of the tectile material revealed the ignition of the untreated sample, while for the fire-retardant – the process of ignition and spread of the flame did not occur. Inhibition of the process of ignition and spread of flame for such a sample is associated with the decomposition of flame retardants under the action of temperature with heat absorption and release of non-combustible gases (nitrogen, carbon dioxide), changing the direction of decomposition towards non-combustible gases and flame retardant coke. This indicates the possibility of transition of the textile material during the processing of the composition to materials that are flammable, do not spread the flame on the surface.

A comparative study between a commercial mixture compound and its individual active ingredients on the cotton leafworm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) on tomatoes under semi-field conditions

BackgroundTomato, Solanum lycopersicum L. (Solanales: Solanaceae) is the second most important vegetable crop in Egypt and is infested with many insect pests. The cotton leafworm, Spodoptera littoralis (Boisd.) causes severe economic losses in tomatoes and many other crops. Many management strategies were developed in order to manage the economic losses obtained. In this context, the present study was conducted to evaluate the effectiveness using a mixture of Emamectin benzoate and lufenuron or using them solely against the 2nd and 4th instar larvae of S. littoralis under semi-field conditions.ResultsThe obtained results showed that the mixture compound show high initial killing effect against 2nd and 4th instar larvae in both growing seasons. Furthermore, the residual effect of the tested compounds also showed the efficiency of the mixture over the solitary active ingredients. In addition, the treatment of the 4th instar larvae with the LC50 of the tested compounds showed significant impacts against the soluble protein, carbohydrate, lipid contents, and the detoxification enzymes.ConclusionIn conclusion, the results showed that the emamectin benzoate and lufenuron could be safe and effective substitute for conventional insecticides either applied solely or in combination.

Various routes of formulated insecticide mixture whole-body acute contact toxicity to honey bees (Apis mellifera)

Honey bees, Apis mellifera (Linnaeus) are crucial pollinators, however large overwinter losses of managed hives and declines in feral colonies have occurred since 2006. Increased insecticide use and the introduction of new insecticide classes have been proposed as factors contributing to these declines. Previous studies have demonstrated contact toxicity of single active ingredient insecticides to honey bees. However, the field realistic contact toxicity of formulated (pre-mix) insecticide mixtures containing multiple active ingredients (each with a different mode of action) to honey bees has not been thoroughly assessed. Here, we simulate whole body contact to spray applications made at low label recommended field rates of thiamethoxam + lambda-cyhalothrin, imidacloprid + beta-cyfluthrin, chlorantraniliprole + lambda-cyhalothrin and methoxyfenozide + spinetoram to honey bees under three realistic scenarios in field crops: (1) continuous exposure, (2) spray-only exposure, and (3) residual exposure. A customized spray tower in a laboratory setting was used to apply insecticides at the lowest recommended dose in the Environmental Protection Agency regulated label of each insecticide. Acute toxicity was determined by screening resulting mortality up to 96-hours post treatment. Mortality varied significantly between treatments at all evaluation periods for all experiments except residual exposure. For continuous and spray-only exposure, thiamethoxam + lambda-cyhalothrin and imidacloprid + beta-cyfluthrin resulted in 80–90% mortality at 48-hours, while at 96-hours we observed close to complete mortality for all treatments. This difference in mortality onset between treatments highlights the importance of extended observation beyond the 48-hours recommended by the USDA-EPA. Our finding of overall high mortality demonstrates the importance of comparing both combined and individual active ingredients in determining insecticide risk to honey bees.

The study of primary DNA damage in the bone marrow of mice under the combined action of pesticides

Introduction. The study of the potential negative effects of combinations of several pesticide active ingredients is an important and understudied area of toxicological and hygienic research. The initial phase of the genotoxicant action on the genetic structures in cells is the primary DNA damage, the identification of which makes it possible to assess the early stages of the genotoxic effect of xenobiotics and their mixtures. The DNA comet assay is widely used for these purposes. The aim of the research is to assess the primary DNA damage under the combined action of pesticides. Materials and methods. To assess DNA damage the experiments on CD-1 mice of both sexes were performed using alkaline comet analysis. The concentration of active products reacting with thiobarbituric acid (TBA) in the blood serum of white outbred rats was assessed as a marker of lipid peroxidation. Results. It was found that mixtures of 2,4-D-acid + glyphosate and thiram + carbendazim did not cause the formation of breaks and alkali-labile sites in the DNA of mice bone marrow cells. Exposure to the combination of the technical grade active ingredients captan and fludioxonil induced the breaks and alkali-labile sites in the DNA of animal bone marrow cells. The comparison of the genotoxicity assessment results obtained by the comet assay and results of analysis of the TBA-active product concentrations in the rat blood serum suggests that the observed primary DNA damage upon exposure to the captan and fludioxonil combination can be mediated by the induction of lipid peroxidation and subsequent interaction of the resulting products with nucleic acids. Conclusion. The results indicate that some pesticides in combination can damage hereditary material in mammalian cells. Therefore, in order to ensure the safe use of pesticides for public health it is necessary to take into account the data on the genotoxicity not only of individual pesticide technical grade active ingredients but also their combinations.

Exposure to multiple pesticides and neurobehavioral outcomes among smallholder farmers in Uganda

BackgroundMultiple epidemiological studies have shown that exposure to single pesticide active ingredients or chemical groups is associated with adverse neurobehavioral outcomes in farmers. In agriculture, exposure to multiple pesticide active ingredients is the rule, rather than exception. Therefore, occupational studies on neurobehavioral effects of pesticides should account for potential co-exposure confounding. MethodsWe conducted a cross-sectional study of 288 Ugandan smallholder farmers between September and December 2017. We collected data on self-reported use of pesticide products during the 12 months prior to survey and estimated yearly exposure-intensity scores for 14 pesticide active ingredients using a semi-quantitative exposure algorithm. We administered 11 neurobehavioral tests to assess five neurobehavioral domains. We implemented a Bayesian Model-Averaging (BMA) approach to examine the association between exposure to multiple pesticides and neurobehavioral outcomes, while accounting for multiple testing. We applied two levels of inference to determine (1) which neurobehavioral outcomes were associated with overall pesticide exposure (marginal inclusion probability (MIP) for covariate-only models <0.5) and (2) which specific pesticide active ingredients were associated with these outcomes (MIP for models where active ingredient was included >0.5). ResultsSeventy-two percent of farmers reported use of pesticide products that contained at least one of 14 active ingredients, while the applicators used in median three different active ingredients (interquartile range (IQR) 4) in the 12 months prior to the study. The most widely used active ingredients were glyphosate (79%), cypermethrin (60%), and mancozeb (55%). We found that overall pesticide exposure was associated with impaired visual memory (Benton Visual Retention Test (BVRT)), language (semantic verbal fluency test), perceptual-motor function (Finger tapping test), and complex attention problems (Trail making A test and digit symbol test). However, when we looked at the associations for individual active ingredients, we only observed a positive association between glyphosate exposure and impaired visual memory (-0.103 [95% Bayesian Credible Interval (BCI)] [-0.24, 0] units in BVRT scores per interquartile range (IQR) increase in annual exposure to glyphosate, relative to a median [IQR] of 6 [3] units in BVRT across the entire study population). ConclusionsWe found that overall pesticide exposure was associated with several neurobehavioral outcome variables. However, when we examined individual pesticide active ingredients, we observed predominantly null associations, except for a positive association between glyphosate exposure and impaired visual memory. Additional epidemiologic studies are needed to evaluate glyphosate’s neurotoxicity, while accounting for co-pollutant confounding.

Whole-Body Acute Contact Toxicity of Formulated Insecticide Mixtures to Blue Orchard Bees (Osmia lignaria).

Blue orchard bees, [Osmia lignaria (Say) (Hymenoptera: Megachilidae)], have been developed as an important pollinator for orchard crops in North America over the last 40 years. The toxicity of several pesticides to O. lignaria and other Osmia species has been previously reported. However, the field-realistic toxicity of formulated premix insecticides comprised of multiple active ingredients (each with a different mode of action) to O. lignaria has not been assessed. Here, we use a customized spray tower in a laboratory setting to assess adult male and female whole-body direct contact exposure to four formulated pesticide mixtures: thiamethoxam + lambda-cyhalothrin (TLC), imidacloprid + beta-cyfluthrin (IBC), chlorantraniliprole + lambda-cyhalothrin (CLC) and methoxyfenozide + spinetoram (MS) by directly spraying anesthetized bees in Petri dishes. Separately, adult male and female whole-body direct contact exposure to formulated imidacloprid (I), beta-cyfluthrin (BC) and their 1:1 binary combination (IBC) was assessed using the same experimental method. Resulting mortality in each study was screened up to 96 h post-treatment to determine acute whole-body contact toxicity. In the first study, TLC and IBC resulted in statistically higher mortality at 24 and 48 h than the two other insecticide combinations tested. The CLC and MS combinations were slower acting and the highest mortality for O. lignaria exposed to these mixtures was recorded at 96 h. We did observe significant differences in toxicity between CLC and MS. In the second study, exposure to the 1:1 binary combination of IBC caused overall significantly higher mortality than exposure to I or BC alone. Both active ingredients alone, however, demonstrated equivalent levels of mortality to the 1:1 binary combination treatment at the 96 h observation reading, indicating increased speed of kill, but not necessarily increased toxicity. Significant differences in the onset of mortality following acute contact whole-body exposure to the formulated insecticide mixtures and individual active ingredients tested were consistently observed across all experiments in both studies.

Occurrence, resistance factors and cross-resistance patterns to herbicides inhibiting acetolactate synthase (ALS) of Echinochloa crus-galli (L.) Pal. Beauv. in Central Europe

Echinochloa crus-galli (L.) Pal. Beauv. is one of the most noxious weeds in maize cultivation and has evolved target-site resistance to ALS-inhibiting herbicides. Due to the hexaploid nature of E. crus-galli, resistance inducing mutations can be harbored by multiple gene copies, but up to now, studies did not include an analysis of the ALS gene copies conferring resistance. Investigations on E. crus-galli populations from different countries in Central Europe revealed the presence of several point mutations conferring resistance and occurring independently at the positions Ala-122, Pro-197 and Trp-574. Moreover, an Asp-376-Glu mutation in the ALS gene in E. crus-galli and a Ser-653-Tyr amino acid change in the ALS protein of a weed were detected for the first time. Additionally, the study revealed the first case of an ALS-resistant population (Trp-574-Leu) in the Czech Republic. The detection of mutations is relevant to the evolution and management of herbicide resistance. Dose–response experiments showed different effects depending on the population and the individual active ingredients and mixtures. Due to the genetic variability of E. crus-galli, results were highly variable. Overall, the populations showed a high level of resistance. For the prevailing mutated positions, i.e., Ala-122, Pro-197 and Trp-574, gene copies were examined separately using molecular genetic methods. A single mutation in at least one out of three ALS gene copies was sufficient to confer resistance at the positions Pro-197 and Trp-574. At Ala-122, point mutations co-occurring in the ALS 1, ALS 2 and ALS 3 gene copy were identified. The study provides a starting point for differentiation of the resistance mechanisms in polyploid E. crus-galli.